ABSTRACT The intestinal lumen, especially that of the distal colon, is occupied by a large and diverse commensal microbiota. On the other hand, the intestinal mucosa contains the largest compartment of the immune system in the body, and these mucosal immune cells must be physically shielded from the dense luminal bacteria. Disruption of the homeostasis between commensal bacteria and mucosal immune cells is recognized as a critical step in the pathogenesis of inflammatory bowl disease (IBD), especially ulcerative colitis (UC). Normally, the luminal bacteria is separated from epithelium and immune cells in the colon by the mucus layer, which consists primarily of mucins that are heavily modified by O-linked oligosaccharides (O-glycans). Over 80% of mucin mass consists of the two types of complex O-glycans, called core 1- and core 3-derived O-glycans. Our published papers in the previous funding period show that mice lacking these complex O-glycans develop spontaneous colitis resembling UC, which indicates O-glycans as crucial components of the mucus barrier. However, how O-glycans maintain mucus barrier function and contribute to the pathogenesis of UC remains to be addressed. In this renewal proposal, we hypothesize that O-glycosylation maintains mucus barrier function by regulating mucin stability, by concentrating and presenting epithelial-derived antimicrobial proteins, and by complementing dietary glycans to prevent microbial dysbiosis. We will test these hypotheses by determining: 1) How mucin O-glycosylation maintains mucus layer homeostasis and barrier function. Sialic acid is a common capping sugar on O-glycans. Some microbiota are known to produce sialidase. In this Aim, we will first test the role of O-glycan sialylation in protecting mucus stability using in vitro mucin degradation assays and a novel mouse line with deficiency of sialylation in intestinal epithelial cells. Then, we will test the role of sialylated O-glycans in the mucus function by concentrating and positioning antimicrobial proteins. 2) How defective O-glycosylation alters mucus/microbiota homeostasis and promotes colitis. In a previous study, we discovered that somatic clonal mutations in the Cosmc gene regulate core 1-O glycosylation in colon epithelial cells of a subset of UC patients, suggesting a partial deficiency of core 1 O-glycans contributes to colitis pathogenesis. First, we will test a hypothesis that combination of a partial loss of core 1 O-glycans, similar to those seen in UC patients, with a polysaccharide-deficient diet, leads to a colitogenic microbiota, which subsequently causes defective mucus barrier and colitis. Second, we will test whether administration of exogenous sialylated oligosaccharides improves mucus barrier function and ameliorates colitis in this model. The mucus layer is an important part of the intestinal barrier, but its biological function is insufficiently studied. The proposed research will determine the contributions of O-glycans, the primary components of the intestinal mucus layer, to intestinal barrier function and pathogenesis of colitis.